Note: Descriptions are shown in the official language in which they were submitted.
t 3386 1 8
VACCINE ADJUVANT
BACKGROUND OF THE INVENTION
Field of the Invention:
This invention relates to improved vaccine adjuvant
formulations, improved processes for preparing said
adjuvant formulations, and methods of using the improved
formulations.
Related Disclosures:
Adjuvants are useful for improving the immune
response obtained with any particular antigen in a
vaccine. Although some antigens are administered in
vaccines without an adjuvant, there are many antigens
that lack sufficient immunogenicity to stimulate a useful
immune response in the absence of an effective adjuvant.
Adjuvants also improve the immune response obtained from
"self-sufficient" antigens, in that the immune response
obtained may be increased or the amount of antigen
administered may be reduced.
The standard adjuvant for use in laboratory animals
is Freund's adjuvant. Freund's complete adjuvant (FCA)
is an emulsion containing mineral oil and killed
mycobacteria in saline. Freund's incomplete adjuvant
(FIA) omits the mycobacteria. Both FIA and FCA induce
exceptional humoral (antibody) immunity, and FCA
additionally induces high levels of cell-mediated
8655Y 26310-FF
*
-2- t 3386 1 8
immunity. However, neither FIA nor FCA are acceptable
for use outside the laboratory due to the adjuvants' side
effects. Mineral oil is known to cause abscesses and
granulomas, while Mycobacterium tuberculosis is the agent
responsible for tuberculosis.
A number of naturally occurring compounds such as
the lipid-A portion of gram negative bacteria endotoxin
and trehalose dimycolate of mycobacteria have been tried
as substitutes for FCA and FIA. Also, the phospholipid
lysolecithin has been shown to have adjuvant activity
(B. Arnold et al., Eur. J. Immunol., 9:363-~66 (1979)).
In addition, several synthetic surfactants, for example,
dimethyldioctadecyl ammonium bromide (DDA) and certain
linear polyoxypropylene-polyoxyethylene (POP-POE) block
polymers (available commercially under the trademark
Pluronic~) have been reported as having adjuvant
activity (H. Snippe et al, Int. Archs. Allergy Appl.
Immun., 65, 390-398 (1981)). R. Hunter et al. have
reported in J. Immunol., 127, 1244-1250 (1981) that
POP-POE block polymers increase antibody formation to
bovine serum albumin (BSA) in mice when used as the
surfactant component of an mineral oil/water emulsion
adjuvant formulation. While these natural and synthetic
surfactants demonstrate some degree of adjuvanticity,
they for the most part fai-l to achieve the degree of
immunopotent~ation obtained using FCA or FIA.
Taking another approach, it has been determined that
the adjuvant effect from mycobacteria is due to a
muramyl-peptide in the cell wall. The smallest fragment
of this molecule that retains adjuvant activity is
N-acetylmuramyl-L-alanyl-D-isoglutamine, commonly known
as muramyldipeptide or "MDP" (Ellouz et al, 8iochem. &
Biophys. Res. Comm., Vol 59, 4, 1317 (1974)). Numerous
analogs of MDP have been prepared, and are also referred
to as "MDPs." See for example Audibert et al., U.S. Pat.
8655Y 26310-FF
~,
1 3386 1 8
--3--
No. 4,158,052; Audibert et al., U.S. Pat. No.
4,220,637; Audibert et al., U.S. Pat. No. 4,323,559;
Baschang et al., U.S. Pat. No. 4,323,560; Baschang et
al., U.S. Pat. No. 4,409,209; Baschang et al., U.S. Pat.
No. 4,423,038; Derrien et al., U.S. Pat. No. 4,185,089;
Hartmann et al., U.S. Pat. No. 4,406,889; Jones et al.,
U.S. Pat. No. 4,082,735; Jones et al., U.S. Pat. No.
4,082,736; Le Francier et al., U.S. Pat. No. 4,427,659;
Le Francier et al., U.S. Pat. No. 4,461,761; Yamamura et
al., U.S. Pat. No. 4,314,998; Yamamura et al., U.S. Pat.
No. 4,101,536; and Yamamura et al., U.S. Pat. No.
4,369,178. While these compounds are weakly effective at
stimulating the immune system when administered in
aqueous solution, the results generally fall short of the
specific immune response obtained with FIA or FCA.
A particularly effective adjuvant formulation
comprising a glycopeptide, a non-toxic POP-POE block
polymer, a glycol ether-based surfactant, a metabolizable
oil, and buffered saline was recently described by
Allison et al., U.S. Pat. No. 4,606,918. This
formulation is capable of inducing strong humoral and
cell-mediated immune responses, equivalent or superior to
the results achieved in laboratory animals using FCA.
However, the formulation is prone to instability and
separation (e.g., creaming) upon standing. We have
discovered that upon refrigeration it loses its ability
to potentiate the primary response to antigens. Also, it
has been found difficult to prepare a stable, homogenous
emulsion with retention of full adjuvant activity on a
commercial scale.
We have now discovered that an immunopotentiating
glycopeptide can be formulated with a non-toxic
N,N,N',N'-tetra(polyoxypropylene-polyoxyethylene)-1,2-
diaminoethane block polymer ("tetra-polyol"), resulting
in an adjuvant emulsion that overcomes certain problems
8655Y 26310-FF
t` 3~38 6 t ~
of the prior art, for example, toxicity and failure to
stimulate cell-mediated immunity. This new adjuvant has
activity equal or greater than the activity of FCA and
Allison's composition. The formulation of the present
invention is easily manufactured with full retention of
activity, and displays greater stability than Allison's
composition. Because the tetra-polyol is non-toxic, this
adjuvant formulation may be safely used as a vehicle for
enhancing the immunogenicity of antigens administered to
birds and mammals.
We have also invented a particularly advantageous
method for preparing adjuvant emulsions, using either
POP-POE block polymers or tetra-polyols, which maintains
the formulations' efficacy, enhances its physical
stability, and reduces its sensitivity to refrigeration.
Remarkably, such adjuvant emulsions may even be frozen
and still retain efficacy.
SUMMARY OF THE INVENTION
One aspect of the invention is an adjuvant in the
form of an emulsion having oily particles dispersed in a
continuous aqueous phase, for potentiating the
immunogenicity of an antigen, which adjuvant comprises:
an emulsion-forming amount of a non-toxic
tetra-polyol or of a POP-POE block polymer; and
an immunopotentiating amount of a glycopeptide;
wherein substantially all of said oily particles
have a diameter less than about 800 nm if a POP-POE block
polymer is present.
Another aspect of the invention is an adjuvant in
the form of an emulsion having oily particles dispersed
in a continuous aqueous phase, for potentiating the
immunogenicity of an antigen, which adjuvant comprises an
emulsion-forming amount of a non-toxic tetra-polyol;
optionally, an emulsion-forming amount of a non-toxic
8655Y 26310-FF
t 3386 1 8
--5--
metabolizable oil; optionally, an emulsion-stabilizing
amount of a glycol ether-based surfactant; water or
aqueous solution, and an immunopotentiating amount of a
muramyldipeptide, preferably a derivative of formula I
f:H2R
~ O H
Rl~ ~ oR3 (I)
R ~ ~HCOR
H ~ ~ X Y
.
and the pharmaceutically acceptable salts thereof,
wherein R and Rl are each independently H or acyl of 1
to 22 carbon atoms, R2 is alkyl or aryl, optionally
substituted with halo, nitro, or lower alkyl, R3 is H,
alkyl, or aryl, R4 is H or lower alkyl, X is L-alanyl,
L- ~aminobutyryl, L-arginyl, L-asparginyl, L-aspartyl,
L-cysteinyl, L-glutaminyl, L-glutamyl, glycyl,
L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl,
L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl,
L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl,
or L-valyl, and Y is D-glutamine, D-isoglutamine or
D-isoasparagine.
Another aspect of the invention is an adjuvant of
the type mentioned above, where an emulsion-forming
amount of a non-toxic POP-POE block polymer may be
substituted for the tetra-polyol, and where substantially
all of the oily particles have a diameter less than about
800 nm, preferably less than 300 nm.
Another aspect of the invention is a vaccine,
comprising an adjuvant of the invention in combination
with an immunogenic amount of an antigen.
8655Y 26310-FF
1 33861 8
Another aspect of the invention is a process for
preparing an adjuvant of the invention, which process
comprises preparing a first mixture comprising the
polymer, oil, surfactant, and water or aqueous solution;
emulsifying the mixture to produce an oil-in-water type
emulsion having oily particles dispersed in a continuous
aqueous phase, wherein substantially all of said oily
particles have a diameter less than about 800 nm,
preferably less than about 300 nm; and combining the
emulsion with a muramyldipeptide derivative of formula I.
Another aspect of the invention is a kit for
extemporaneous preparation of an adjuvant of the
invention, which kit comprises: a first container
containing an emulsion as described above, and a second
container containing the muramyl dipeptide, preferably
N-acetylmuramyl-L-threonyl-D-isoglutamine optionally in
an aqueous solution or suspension, where the
concentrations of the components in each container are
selected such that combination of the contents of both
containers produces an adjuvant of the invention.
Another aspect of the invention is a kit for the
preparation of a vaccine of the invention, which differs
from the adjuvant kit described above in that an
immunogenic amount of an antigen is added to the second
container, or present in a third container.
Another aspect of the invention is a method for
inducing an immune response in an animal having an immune
system, which method comprises administering a vaccine of
the invention.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS
One aspect of the invention is an adjuvant in the
form of an emulsion having oily particles dispersed in a
continuous aqueous phase, for potentiating the
immunogenicity of an antigen, which adjuvant comprises:
8655Y 26310-FF
1 3386 1 8
--7--
an emulsion-forming amount of a non-toxic
tetra-polyol or of a POP-POE block polymer; and
an immunopotentiating amount o~ a glycopeptide;
wherein substantially all of said oily particles
have a diameter less than about 800 nm if a POP-POE block
polymer is present.
Another aspect of the invention is an adjuvant in
the form of an emulsion having oily particles dispersed
in a continuous aqueous phase, for potentiating the
immunogenicity of an antigen, which adjuvant comprises an
emulsion-forming amount of a non-toxic tetra-polyol;
optionally, an emulsion-forming amount of a non-toxic
metabolizable oil; optionally an emulsion-stabilizing
amount o~ a glycol ether-based sur~actant; water or
aqueous solution, and an immunopotentiating amount of a
muramyldipeptide, preferably a derivative o~ ~ormula I
~H2R
Rl~ ~ ~ oR3 (I)
R4 ~HCOR2
~r~
H ~ X-~
and the pharmaceutically acceptable salts thereof,
wherein R and R1 are each independently H or acyl of 1
to 22 carbon atoms, R2 is alkyl or aryl, optionally
substituted with halo, nitro, or lower alkyl, R3 is H,
alkyl, or aryl, R4 is H or lower alkyl, X is L-alanyl,
L-~-aminobutyryl, L-arginyl, L-asparginyl, L-aspartyl,
L-cysteinyl, L-glutaminyl, L-glutamyl, glycyl,
L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl,
L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl,
8655Y 26310-FF
1 3386 1 8
--8--
L-prolyl, L-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl,
or L-valyl, and Y is D-glutamine, D-isoglutamine or
D-isoasparagine. A preferred subgenus is the adjuvant
wherein said tetra-polyol is Tetronic~ 1501,
particularly where said muramyldipeptide derivative of
formula I is N-acetylmuramyl-L-threonyl-D-isoglutamine.
A preferred class of the invention is the adjuvant which
includes a non-toxic metabolizable oil, especially where
said oil is squalene or squalane. A preferred subclass
is the adjuvant wherein said glycol ether-based
surfactant is Tween~ 80, particularly where said water
or aqueous solution comprises isotonic buffered saline,
and especially where substantially all of the oily
particles have a diameter less than about 800 nm,
preferably less than about ~00 nm. Another preferred
subgenus is the adjuvant wherein said tetra-polyol is
Tetronic~ 1501 and said muramyldipeptide derivative of
formula I is murabutide.
Another aspect of the invention is an adjuvant in
the form of an oil-in-water type emulsion, having oily
particles dispersed in a continuous aqueous phase, for
potentiating the immunogenicity of an antigen, which
adjuvant comprises a non-toxic tetra-polyol in an amount
f 0.2 to 49%; a non-toxic metabolizable oil in an
amount of 0-15X; a glycol ether-based surfactant in an
amount of 0.05-5%; water or aqueous solution; and
0.0001-10% of a muramyldipeptide derivative of formula I
(% are vol./vol., except for the muramyldipeptide which
is wt./vol.). A preferred subgenus is the adjuvant
- wherein said tetra-polyol is Tetronic~ 1501,
particularly where said muramyldipeptide derivative of
formula I is N-acetyl-muramyl-~-threonyl-D-isoglutamine.
A preferred class is the adjuvant which includes a
non-toxic metabolizable oil, wherein said oil is squalene
or squalane. A presently preferred embodiment of the
8655Y 26~10-FF
1 3386 1 8
_9_
invention is the adjuvant in the form of an oil-in-water
type emulsion, having oily particles dispersed in a
continuous aqueous phase, for potentiating the
immunogenicity of an antigen, which adjuvant comprises
Tetronic~ 1501 in an amount of 1-10%; squalane or
squalene in an amount of 1-10%; Tween~ 80 in an amount
of about 0.2X; isotonic buffered saline (using phosphate
buffers, acetate buffers, or combinations thereof); and
O O.OOOl-lOX N-acetylmuramyl-L-threonyl-D-isoglutamine,
particularly where substantially all of said oily
particles have a diameter less than about 800 nm,
preferably less than about 300 nm.
Another aspect of the invention is an adjuvant in
the form of an oil-in-water type emulsion, having oily
particles dispersed in a continuous aqueous phase, for
potentiating the immunogenicity of an antigen, which
adjuvant comprises an emulsion-forming amount of a
non-toxic POP-POE block polymer; optionally, an
emulsion-forming amount of a non-toxic metabolizable
oil; an emulsion-stabilizing amount of a glycol
ether-based surfactant; water or aqueous solution; and
an immunopotentiating amount of a muramyldipeptide
derivative of formula I, wherein substantially all of
said oily particles have a diameter less than about
800 nm, preferably less than about 300 nm. A preferred
subgenus is the adjuvant wherein said non-toxic POP-POE
block polymer is Pluronic~ L121, particularly where
said muramyldipeptide derivative of formula I is N-acetyl-
muramyl-L-threonyl-D-isoglutamine. A preferred class is
the adjuvant which includes a non-toxic metabolizable
oil, wherein said oil is squalene or squalane. A
preferred subclass is the adjuvant wherein said glycol
ether-based surfactant is Tween~ 80, particularly where
said water or aqueous solution comprises isotonic
buffered saline.
8655Y 26310-FF
~,,r
1 3386 1 8
--10--
Another aspect of the invention is an adjuvant in
the form of an oil-in-water type emulsion, having oily
particles dispersed in a continuous aqueous phase, for
potentiating the immunogenicity of an antigen, which
adjuvant comprises a non-toxic POP-POE block polymer in
an amount of 0.2 to 49X; a non-toxic metabolizable oil
in an amount of 0-15%; a glycol ether-based surfactant
in an amount of 0.05-5X; water or aqueous solution; and
O.OOOl-lOg of a muramyldipeptide derivative of formula I,
where substantially all of said oily particles have a
diameter less than about 800 nm, preferably less than
about 300 nm (X are vol./vol., except for the
muramyldipeptide which is wt./vol.). A preferred
subgenus is the adjuvant wherein said POP-POE block
polymer is Pluronic~ L121, particularly where said
muramyldipeptide derivative of formula I is
N-acetylmuramyl-L-threonyl-D-isoglutamine. A preferred
class is the adjuvant which includes a non-toxic
metabolizable oil, wherein said oil is squalene or
squalane. A preferred subclass is the adjuvant wherein
said glycol ether-based surfactant is Tween~ 80. A
presently preferred embodiment is the adjuvant in the
form of an oil-in-water type emulsion, having oily
particles dispersed in a continuous aqueous phase, for
potentiating the immunogenicity of an antigen, which
adjuvant comprises Pluronic~ L121 in an amount of
l-lOX; squalane or squalene in an amount of 1-10%;
Tween~ 80 in an amount of about 0.2X; isotonic
buffered saline; and 0.0001-10% N-acetylmuramyl-L-
threonyl-D-isoglutamine, wherein substantially all of
said oily particles have a diàmeter less than about
800 nm, preferably less than about 300 nm.
Another aspect of the invention is a vaccine
comprising an adjuvant of the invention in combination
with an immunogenic amount of an antigen. Suitably this
8655Y 26310-FF
i..
t 3386 1 8
--11--
is a vaccine in the form of an oil-in-water type
emulsion, having oily particles dispersed in a continuous
aqueous phase, for immunizing an animal, which vaccine
comprises an immunogenic amount of an antigen; an
emulsion-forming amount of a non-toxic tetra-polyol or a
non-toxic POP-POE block polymer; optionally, an
emulsion-forming amount of a non-toxic metabolizable oil;
optionally an emulsion-stabilizing amount of a glycol
ether-based surfactant; water or aqueous solution; and
an immunopotentiating amount of a muramyldipeptide,
preferably a derivative of formula I. A preferred
subgenus is the vaccine which includes a tetra-polyol,
especially where said tetra-polyol is Tetronic~ 1501.
A preferred class is the vaccine wherein substantially
all of said oily particles have a diameter less than
about 800 nm, preferably less than about 300 nm. A
preferred subclass is the vaccine wherein said
muramyldipeptide derivative of formula I is N-acetyl-
muramyl-L-threonyl-D-isoglutamine. Another preferred
subclass is the vaccine wherein said muramyldipeptide
derivative of formula I is murabutide. A presently
preferred embodiment is the vaccine which comprises:
Tetronic~ 1501 in an amount of 1-10%; squalane or
squalene in an amount of l-lOX; Tween~ 80 in an amount
of about 0.2%; isotonic buffered saline; and 0.0001-10%
N-acetylmuramyl-L-threonyl-D-isoglutamine, especially
where substantially all of said oily particles have a
diameter less than about 800 nm, preferably less than
about 300 nm. Another preferred subgenus is the vaccine
which includes a POP-POE block polymer, wherein said
block polymer is Pluronic~ L121, wherein substantially
all of said oily particles have a diameter less than
about 800 nm, preferably less than about 300 nm. A
preferred class is the vaccine wherein said muramyl-
dipeptide derivative of formula I is N-acetylmuramyl-L-
8655Y 26310-FF
~.
-12- 1 3386 1 8
threonyl-D-isoglutamine. Another preferred class is the
vaccine wherein said muramyldipeptide derivative of
formula I is murabutide. A presently preferred
embodiment is the vaccine which comprises: Pluronic~
121 in an amount of 1-10%; squalane or squalene in an
amount of l-lOX; Tween~ 80 in an amount of about
0.2%; isotonic buffered saline; and O.OOOl-lOX N-acetyl-
muramyl-L-threonyl-D-isuglutamine.
Another aspect of the invention is a process for
preparing the adjuvant or vaccine of the invention, which
process comprises mixing together the aqueous phase and
the emulsion-forming amount of the non-toxic tetra-polyol
or of the POP-POE block polymer so as to form an emulsion.
Another aspect of the invention is a process for
preparing an adjuvant of the invention, which process
comprises: preparing a first mixture comprising a
non-toxic tetra-polyol or a non-toxic POP-POE block
polymer, optionally a non-toxic metabolizable oil,
optionally a glycol ether- based surfactant, and water or
aqueous solution; emulsifying said first mixture to
produce an oil-in-water type emulsion, having oily
particles dispersed in a conti~uous aqueous phase,
wherein substantially all of said oily particles have a
diameter less than about 800 nm, preferably less than
about 300 nm; and combining said emulsion with a
muramyldipeptide derivative of formula I. A preferred
class is the process wherein said first mixture is
emulsified using a Microfluidizer~ (or other suitable
emulsifying technique) to obtain an emulsion wherein
substantially all of said oily particles have a diameter
less than about 800 nm, preferably less than about
300 nm. A preferred subclass is the process wherein said
muramyldipeptide derivative of formula I is combined with
said emulsion in the form of an aqueous solution or
35 suspension.
8655Y 26310-FF
1 3386 1 8
-13-
Another aspect of the invention is a kit for
extemporaneous preparation of an adjuvant of the
invention, which kit comprises:
a first container containing the emulsion of the
tetra-polyol or POP-POE polymer in the aqueous phase; and
a second container containing the glycopeptide.
Another aspect of the invention is a kit for
extemporaneous preparation of a vaccine of the invention,
which kit comprises:
a first container containing the emulsion of the
tetra-polyol or POP-POE block polymer in the aqueous
phase; and
a second container containing the antigen;
wherein the glycopeptide may be present in a third
container, or in the first or second containers.
Another aspect of the invention is a kit for
extemporaneous preparation of an adjuvant of the
invention, which kit comprises: a first container
containing an oil-in-water type emulsion, having oily
particles dispersed in a continuous aqueous phase, where
said emulsion comprises Tetronic~ 1501 or Pluronic~
L121, squalane or squalene, optionally Tween ~80, and
isotonic buffered saline; and a second container
containing N-acetylmuramyl-L- threonyl-D-isoglutamine in
powder form (preferably lyophilized) or in aqueous
solution or suspension, where the concentrations of the
components in each container are selected such that
combination of the contents of both containers produces a
formulation comprising Tetronic~ 1501 or Pluronic ~
L121 in an amount of 1-30%, squalane or squalene in an
amount of 1-30X, optionally Tween~ 80 in an amount of
about 0.2-5%, 0.0001-30% N-acetylmuramyl-L-threonyl-D-
isoglutamine, and isotonic buffered saline. A preferred
subgenus is the kit wherein Tetronic ~1501 is
included. A preferred class is the kit wherein
8655Y 26310-FF
~r
1 3386 1 8
-14-
substantially all of said oily particles have a diameter
less than about 800 nm, preferably less than about
~00 nm. Another preferred subgenus is the kit wherein
Pluronic~ L121 is included.
Another aspect of the invention is a kit for
extemporaneous preparation of a vaccine of the invention,
which kit comprises: a first container containing an
oil-in-water type emulsion, having oily particles
dispersed in a continuous aqueous phase, where said
emulsion comprises Tetronic~ 1501 or Pluronic~ L121,
squalane or squalene, optionally Tween~ 80, and
isotonic buffered saline; and a second container
containing N-acetylmuramyl-L-threonyl-D- isoglutamine in
powder form (preferably lyophilized) or in aqueous
solution or suspension and an immunogenic amount of an
antigen; where the concentrations of the components in
each container are selected such that combination of the
contents of both containers produces a formulation
comprising Tetronic~ 1501 or Pluronic~ L121 in an
amount of l-lOX, squalane or squalene in an amount of
1-10%, optionally Tween~ 80 in an amount of about 0.2%,
O.OOOl-lOX N-acetylmuramyl-L-threonyl-D-isoglutamine, an
immunogenic amount of an antigen, and isotonic buffered
saline. Optionally, the antigen can be in a separate
third container. A preferred subgenus is the kit wherein
Tetronic~ 1501 is included. A preferred class is the
kit wherein substantially all of said oily particles have
a diameter less than about 800 nm, preferably less than
about 300 nm. Another preferred subgenus is the kit
wherein Pluronic~ L121 is included.
As noted, suitably in the kits of the invention the
glycopeptide is present as a powder, preferably a
lyophilized powder.
Another aspect of the invention is a method for
inducing an immune response in an animal having an immune
8655Y 26310-FF
1 3386 1 8
system, which method comprises: administering a vaccine
comprising Pluronic~ L121 or Tetronic~ 1501 in an
amount of 1-10%; squalane or squalene in an amount of
1-10%; optionally Tween~ 80 in an amount of about
0.2%; isotonic buffered saline; 0.0001-10
N-acetylmuramyl-L-threonyl-
D-isoglutamine; and an immunogenic amount of an antigen.
DEFINITIONS
The term "alkyl~ refers to a straight or branched
radical comprised of 1 to 22 carbon atoms containing no
unsaturation. Examples of alkyl are methyl, ethyl,
propyl, butyl, tert-butyl, hexyl, octyl, decyl, dodecyl,
eicosanyl, and the like. "Lower alkyl" refers to an
alkyl radical of 1 to 7 carbon atoms, for example,
methyl, ethyl, propyl, butyl, tert-butyl, hexyl, 3-methyl-
hexyl, heptyl, and the like. "Cycloalkyl" refers to
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, and the like.
The term "acyl" refers to radicals of the formula
RCO-, where R is H or alkyl as defined above. "Lower
acyl" refers to such radicals where R is H or lower
alkyl. Examples of acyl include formyl, acetyl,
propionyl, butyryl, pentanoyl, hexanoyl, eicosanoyl, and
the like. Examples of lower acyl include formyl, acetyl,
propionyl, butyryl, pentanoyl, hexanoyl, and the like.
The term ~halo" as used herein refers to fluoro,
chloro, bromo and iodo.
The term "alkoxy" refers to a radical of the form
RO-, where R is lower alkyl or cycloalkyl as defined
above.
The term "aryl" refers to aromatic radicals
consisting entirely of carbon and hydrogen, containing
from 6 to 12 carbon atoms. Examples of aryl groups are
phenyl, naphthyl, and the like.
8655Y 26~10-FF
.~ ~
1 33861 8
-16-
The term "pharmaceutically acceptable salts" refers
to acid addition salts of the subject compounds which
possess the desired pharmacological activity and which
are neither biologically nor otherwise undesirable.
These salts are formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid,
nitric acid or phosphoric acid; or organic acids such as
acetic acid, propionic acid, glycolic acid, pyruvic acid,
malonic acid, succinic acid, malic acid, maleic acid,
fumaric acid, tartaric acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid and the like.
The term "treatment" as used herein covers any
treatment of a disease in a bird or mammal, particularlY
a human, and includes:
(i) preventing the disease from occurring in a
subject which may be predisposed to the disease but has
not yet been diagnosed as having it;
(ii) inhibiting the disease, i.e., arresting its
development; or
(iii) relieving the disease, i.e., causing
regression of the disease. (It should be noted that
vaccination may effect regression of a disease where the
disease persists due to ineffective antigen recognition
by the subject's immune system, where the vaccine
effectively presents antigen.)
The term "optionally substituted" as applied to aryl
radicals in the invention means that the radical may be
unsubstituted or substituted with one to three halo,
nitro, lower alkyl, or lower alkoxy groups. The optional
substituents may be the same or different.
The term "muramyl dipeptide derivative" includes
compounds of formula I:
8655Y 26~10-FF
-17- t 33861 8
2R
~ 0 H
R1' ~ oR3 (I~
~,~
~ ~Y
where ~, Rl, R2, R3, and R4 are each
independently H, alkyl, acyl, or aryl optionally
substituted with halo, nitroJ or lower alkyl; X is one
or several amino acids, and Y is D-glutamine,
D-isoglutamine or D-lsoasparag$ne, which may optionally
be esterified or amidated Pre~erred compounds are those
o~ formula 1 ~herein R and Rl are H or acyl of l to 22
car~on atoms; R2 is methyl; R3 is hydrogen; X is
L-alanyl, L- ~amino~utyryl, L-arginyl, L-asparginyl,
L-aspartyl, L-cyste$nyl, L-glutaminyl, L-glutamyl,
glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl,
L-leucyl, L-lysyl, L-methlonyl, L-ornithinyl,
L-phenylalanyl, L-prolyl, L-seryl, L-threonyl, L-tyrosyl,
L-tryptophanyl, or L-valyl, and Y is D-glutamine or
0-isoglutamine. The most pre~erred MDPs are N-acetyl-
muramyl-L- ~aminobutyryl-D-isoglutamine; 6-0-stearoyl-
N-acetylmuramyl-L- ~amino~utyryl-0-$soglutamine;
N-acetylmuramyl-L-threonyl-D-isoglutamine; N-acetyl-
muramyl-L-valyl-0-isoglutamine; N-acetylmuramyl-L-alanyl-
D-isoglutamine; N-acetyl-desmethylmuramyl-L-alanyl-D-
isoglutamine; N-acetylmuramyl-L-alanyl-D-glutamine ~utyl
ester (murabutide); N-acetylmuramyl-L-seryl-
D-isoglutamine; and N-butyrylmuramyl-L-( ~aminobutyryl)-
0-isoglutamine Another useful MDP is N-acetyl-
(n-~utylmuramyl)-L- ~aminobutyryl-D-isoglutamine
8655Y 26~10-FF
1 3386 1 8
-18-
The term "immunopotentiating amount" refers to the
amount of MDP derivative needed to effect an increase in
antibody titer and/or cell mediated immunity when
administered with an antigen in the formulation of the
S invention, as compared with the titer level observed in
the absence of the MDP. As can be appreciated, each MDP
may have an effective dose range that may differ from
other MOPs. Thus, a single dose range cannot be
prescribed which will have a precise fit for each
possible glycopeptide within the scope of this
invention. However, the immunopotentiating amount may
easily be determined by one of ordinary skill in the
art. As a general rule, the glycopeptide will preferably
be present in an amount of between 0.001 and 2X. A more
preferred amount is 0.005 to lX.
The term "non-toxic metabolizable oil" refers to an
oil of 6 to 30 carbon atoms including, but not limited
to, alkanes, alkenes, alkynes, and their corresponding
acids and alcohols, the ethers and esters thereof, and
mixtures thereof. The oil may be any vegetable oil, fish
oil, animal oil or synthetically prepared oil which can
be metabolized in the body of the subject to which the
adjuvant is administered, and which is not toxic to the
organism. It is essential that the oil be metabolized by
the animal or bird to which it is administered to avoid
causing abscesses, granulomas or carcinomas. Nuts, seeds
and grains are common sources of vegetable oils.
Synthetic oils within the scope of this invention include
"Neobee~n (available from PVO International, Inc.,
Chemical Specialities Division, 416 Oivision Street,
Boongon, New Jersey) and others. Shark liver oil
contains a branched, unsaturated oil known as squalene,
2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa-
hexene which is particularly preferred herein. Squalane,
the saturated analog of squalene is also a particularly
8655Y 26310-FF
~r
-19- 1 3386 1 8
preferred ail. Fish oils, including squalene and
squalane, are readily available from commercial sources
or may be obtained by methods known in the art.
An "emulsion-forming amount" of a non-toxic
metabolizable oil is that amount which will form an
emulsion in the presence of the tetra-polyol or POP-POE
block polymer. The oil component of these adjuvants and
vaccine formulations will usually be present in an amount
between 1 to 30%, but preferably in an amount of 1 to
lOX. It is most preferred to use about a 5%
concentration of oil.
The term "substantially all" as applied to the size
of the oily emulsion particles means that greater than
70% of the particles are below the size stated (e.g.,
800 nm or 300 nm), preferably 80% of the particles, and
most preferably 95% or more.
The aqueous portion of these adjuvant compositions
is preferably buffered isoosmotic saline. Because these
compositions are intended for parenteral administration,
it is preferred to formulate these solutions so that the
tonicity is essentially the same as normal physiological
fluids in order to prevent post-administration swelling
or rapid absorption of the composition due to
differential ion concentrations between the composition
and physiological fluids. It is also preferred to buffer
the saline in order to maintain a pH compatible with
normal physiological conditions. Also, in certain
instances, it may be necessary to maintain the pH at a
particular level in order to insure the stability of
certain composition components, such as the
glycopeptides. Any physiologically acceptable buffer may
be used herein, but it has been found that it is most
convenient to use a phosphate buffer. Any other
acceptable buffer such as acetate, Tris, bicarbonate,
carbonate, and the like can be used as a substitute for a
8655Y 26310-FF
1 33861 8
-20-
phosphate buffer. It is preferred to use phosphate
buffered saline, or saline buffered with a mixture of
phosphate and acetate.
The term ~antigen" refers to any substance, usually
a protein or glycoprotein, lipoprotein, saccharide,
polysaccharide or lipopolysaccharide, which upon
administration stimulates the formation of specific
antibodies and reacts specifically in vivo or in vitro
with a homologous antibody. Moreover, it stimulates the
proliferation of T-lymphocytes with receptors for the
antigen, and can react with the lymphocytes to initiate
the series of responses designated cell-mediated immunity.
The term "antigen" as used herein also includes
combinations of haptens with a carrier. A hapten is a
portion of an antigenic molecule or antigenic complex
that determines its immunological specificity, but is not
sufficient to stimulate an immune response in the absence
of a carrier. Commonly, a hapten is a relatively small
peptide or polysaccharide and may be a fragment of a
naturally occurring antigen. In artificial antigens, it
may be a low molecular weight substance such as, for
example, an arsanilic acid derivative. A hapten will
react specifically in vivo and in vitro with homologous
antibodies or T-lymphocytes. Haptens are typically
attached to a large carrier molecule such as bovine serum
albumin (9SA) or keyhole limpet hemocyanin (KLH) by
either covalent or non-covalent binding before
formulation as a vaccine. For example, a common
artificial antigen used to test vaccines and adjuvants
consists of 2,4-dinitrophenol (DNP) covalently bound to
BSA. Suitable antigens for use in this invention include
hepatitis B (surface) antigen, and influenza (for
example, A or 8) antigen. Also, antigens for AIDS and
herpes.
8655Y 26310-FF
-21- 1 3386 1 8
The term "immunogenic amount" of an antigen refers
to an amount of antigen sufficient to stimulate a useful
immune response, when administered with an adjuvant of
the invention. The amount of antigen necessary to
provide an immunogenic amount is readily determined by
one of ordinary skill in the art, e.g., by preparing a
series of vaccines of the invention with varying
concentrations of antigen, administering the vaccines to
suitable laboratory animals (e.g., Guinea pigs), and
assaying the resulting immune response by measuring serum
antibody titer, antigen-induced swelling in the skin, and
the like.
The term "tetra-polyol" as used herein refers to
N,N,N',N'-tetra(polyoxypropylene-polyoxyethylene)-1,2-
diaminoethane block polymers. These compounds may beprepared hy the process disclosed in U.S. Patent No.
2,979,528, or may be obtained commercially from
BASF-Wyandotte under the trademark Tetronic~.
Tetronic~ polyols are designated with a three or
four digit number which indicates the average molecular
weight of the polyoxypropylene (POP) portion and the
percentage of the total molecular weight contributed by
the polyoxyethylene (POE) portion of the molecule. The
first one or two non-zero digits indicate the average
molecular weight of the POP section, ranging from
501-1000 for Tetronic~ 304 to 6500-7000 for TetroniC~
1501. The last digit indicates the percentage of POE in
10% increments, ranging from lOX for Tetronic~ 1501 to
80% for Tetronic~ 1508. The characteristics of these
compounds are determined by the molecular weight of the
POP portion and the amount of POE in the product.
Preferred tetra-polyols in the practice of the invention
are relatively insoluble in water at 25C and have low
HLB values. The HLB value should preferably be lower
than about 5Ø Presently preferred tetra-polyols are
8655Y 26~10-FF
-~r
t 3386 1 8
-22-
Tetronic~ 1501, Tetronic~ 1~01, Tetronic~ 1101, and
Tetronic~ 1502, particularly Tetronic~ 1501 and
Tetronic~ 1~01, especially Tetronic~ 1501. Other
appropriate tetra-polyols with the necessary properties
may be prepared using the methods disclosed in U.S.
Patent No. 2,979,528, and are to be considered
equivalents within the scope of this invention. For
example, one could prepare a tetra-polyol wlth a POP
molecular weight of 8,000 and a POE content of 8%. The
properties necessary are (i) low HLB value (< 5.0,
preferably < 2.0); (ii) little or no aqueous
solubility; (iii) forms stable emulsions with the
addition of a glycol ether-based surfactant; and (iv)
lack of toxicity.
The term "POP-POE block polymer" refers to a polymer
made by the sequential addition of propylene oxide and
then ethylene oxide to a low molecular weight, reactive
compound, usually propylene glycol. These block polymers
can be prepared by the methods set out in U.S. Patent
2,674,619 issued to Lunsted, and are commercially
available from BASF-Wyandotte under the trademark
Pluronic~. The characteristics of these polyols are
determined by the molecular weight of the POP nucleus and
of the percentage POE in the product. The POP section
imparts hydrophobic characteristics to the block polymer,
while the POE section imparts hydrophilic
characteristics. Preferred block polymers are determined
by the same criteria used to select appropriate
tetra-polyols. Preferred block polymers for the practice
of the invention are Pluronic~ L121 and L101.
Pluronic~ polyols are designated by a letter
prefix followed by a two or a three digit number. The
letter prefixes (L, P, or F) refer to the physical form
of each polymer, (liquid, paste, or flakeable solid).
The first one or two digits is a code for the average
8655Y 26310-FF
1 3386 1 8
molecular weight of the POP base, while the last digit
indicates the amount of POE. For example, Pluronic~
L101 is a liquid having a polyoxypropylene base of
average molecular weight 3,250, with 10% polyoxyethylene
present at the ends of the molecule. The preferred block
polymers are those which are liquid over a temperature
range between about 15-40C. In addition, polymer
mixtures of liquid and paste, liquid, paste and flakeable
solid or liquid and flakeable solid mixtures which are
liquid within the specified temperature range may have
utility in this invention.
Preferred block polymers are those having a POP base
ranging in molecular weight between about 2250 and 4300
and POE in an amount between about 1 and ~OX. More
preferred are those polymers wherein POP has a molecular
weight falling between ~250 and 4000 and the POE
component comprises 10-20X. The Pluronic~ polyols
L101, L121 and L122 fall within this definition. Most
preferred are the polymers wherein POP has a molecular
weight of 4000 and POE in an amount of 10% or POP has a
molecular weight of 3250 and POE in an amount of 10% eg.
Pluronic~ polyols L121 and L101 respectively.
An "emulsion-forming amount" of tetra-polyol or
POP-POE block polymer is that quantity which will form
micelles or an emulsion. For the purposes of the
invention this is an amount between 0.2% and 49X by
volume. A more preferred amount is from 0.2% to 20%, and
about 1-5% is even more preferred. A concentration of
1-2.5% is presently most preferred.
The term "surfactant" refers to non-toxic surface
active agents capable of stabilizing the emulsion. There
are a substantial number of emulsifying and suspending
agents generally used in the pharmaceutical sciences.
These include naturally derived materials such as gums,
vegetable protein, alginates, cellulose derivatives,
8655Y 26~10-fF
~r
t 3386 1 8
-24-
phospholipids (whether natural or synthetic), and the
like. Certain polymers having a hydrophilic substituent
on the polymer backbone have surfactant activity, for
example, povidone, polyvinyl alcohol, and glycol
ether-based compounds. Compounds derived from long chain
fatty acids are a third substantial group of emulsifying
and suspending agents usable in this invention. Though
any of the foregoing surfactants can be used so Long as
they are non-toxic, glycol ether-based surfactants are
preferred. Preferred surfactants are non-ionic. These
include polyethylene glycols (especially PEG 200, 300,
400, 600 and 900), Span~, Arlacel~, Tween~,
Myrj~, Brij~ (all available from ICI, America's Inc.,
Wilmington, Delaware), polyoxyethylene, polyol fatty acid
esters, polyoxyethylene ether, polyoxypropylene fatty
ethers, bee's wax derivatives containing polyoxyethylene,
polyoxyethylene lanolin derivatives, polyoxyethylene
fatty glycerides, glycerol fatty acid esters or other
polyoxyethylene acid alcohol or ether derivatives of
long-chain fatty acids of 12-21 carbon atoms. The
presently preferred surfactant is Tween~ 80 (otherwise
known as polysorbate 80 for polyoxyethylene 20 sorbitan
monooleate), although it should be understood that any of
the above-mentioned surfactants would be suitable after
lack of toxicity is demonstrated.
An ~emulsion-stabilizing amount of a glycol ether-
based surfactant~ is usually effected by having the
surfactant present in an amount of 0.05 to 5%. An amount
of 0.2X to lX is preferred.
PREPARATION
The components of the adjuvant of the invention may
be obtained through commercial sources, or may be
prepared by one of ordinary skill in the art.
8655Y 26310-FF
1 ~3~
-25-
The tetra-polyols may be prepared by the process
disclosed in U.S. Patent No. 2,979,528, or may be
obtained commercially from BASF-Wyandotte under the
trademark Tetronic~.
The POP-POE block polymers can be prepared by the
methods set out in U.S. Patent 2,674,619 issued to
Lunsted, and are commercially available from 8~SF-
Wyandotte under the trademark Pluronic~.
The glycol-ether based surfactants PEG 200, 300,
400, 600 and 900, Span~, Arlacel~, Tween~, Myrj~,
Brij~, and the like are readily available commercially
from ICI, America's Inc., Wilmington, Delaware, and
others.
The non-toxic metabolizable oils are available from
a variety of sources: e.g., squalane and squalene are
available from Aldrich Chemical Co.
The MDPs may be obtained commercially from sources
such as Sigma ~hemical Co., or prepared following the
processes disclosed in Audibert et al., U.S. Pat. No.
4,158,052; Audibert et al., U.S. Pat. No. 4,220,637;
Audibert et al., U.S. Pat. No. 4,323,559; ~aschang et
al., U.S. Pat. No. 4,323,560; Baschang et al., U.5. Pat.
No. 4,409,209; Paschang et al., U.S. Pat. No.
4,423,038; ~errien et al., U.S. Pat. No. 4,185,089;
Hartmann et al., U.S. Pat.-No. 4,406,889; Jones et al.,
U.S. Pat. No. 4,082,735; Jones et al., U.S. Pat. No.
4,082,736; Le Francier et al., U.S. Pat. No. 4,427,659;
Le Francier et al., U.S. Pat. No. 4,461,761; Yamamura et
al., U.S. Pat. No. 4,314,998; Yamamura et al., U.S. Pat.
No. 4,101,536; and Yamamura et al., U.S. Pat. No.
4,369,178, all incorporated herein by reference.
The adjuvant is prepared by emulsification, using a
mixer. If the adjuvant is to be prepared on a laboratory
scale using a tetra-polyol for immediate use, it may be
35 mixed simply by hand. For example, Tween~ 80 and
8655Y 26310-F~ -
1 33 8 6 1 8
- 26 -
buffered saline are added to squalane and Tetronic~ 1501 in a
test tube at 2X concentration, and the combination mixed
using a vortex mixer to form an emulsion. To this is added a
2X solution of antigen and MDP in buffered saline to form the
completed vaccine. It is more preferred to use a high-shear
mixer such as a Greerco Homogenizer Mixer to form a smoother,
more homogenous emulsion.
Preferably, the adjuvant emulsion is "microfluidized"
prior to adding the antigen, whether a tetra-polyol or a POP-
POE block polymer is used. This is accomplished using a very
high-shear mixer such as a Microfluidizer~ (commercially
available through Microfluidics Corp., Newton, MA). With the
Microfluidizer~, typically 100-500 mL batches of emulsion are
prepared. The emulsion is cycled through the Microfluidizer~
about 2-10 times, until the emulsion particle size reaches
the desired level, preferably a diameter less than 800 nm,
preferably less than about 300 nm, most preferably, less than
about 200 nm. The Microfluidizer~ combines shear, turbulence
and cavitation forces, the two fluidized streams interacting
at very high velocities within an interaction chamber thus
creating uniformly small emulsion particles. It should be
understood that equipment other than the Microfluidizer~ may
be capable of producing a satisfactory emulsion, and that the
use of any device capable of producing an emulsion of
sufficient stability and sufficiently small particle size is
within the scope of this process. Normally, the MDP will be
added after the emulsification step. If the emulsion is to
be stored prior to the addition of the MDP and the antigen,
this may be done with refrigeration and/or under nitrogen.
The resulting emulsion may be assayed in a variety of
ways well known in the art. The emulsion stability may be
measured by allowing the emulsion to stand at room
temperature, and under refrigeration, followed by observation
for separation into phases. this assay may be accelerated by
centrifuging the emulsion, e.g., for two hours at 4500g.
1 3386 1 8
-27-
Particle size may be determined by, for example,
optical microscopy, transmission electron microscopy, and
laser light-scattering techniques, preferably using laser
photon correlation spectroscopy (PCS). PCS analysis may
be performed using, for example, a Nicomp Model 200 laser
particle sizer, with a model TC-lOO computing
autocorrelator.
Biological activity may be assayed using standard
laboratory techniques, e.g., by vaccinating a standard
laboratory animal (e.g., a Guinea pig) with a standard
antigen (e.g., BSA or DNP-BSA) using a test adjuvant
formulation. After allowance of time for boosting the
vaccination, and time for immunization to occur, the
animal is challenged with the standard antigen and the
results measured. The response may be quantified by any
measure accepted in the art for measuring immune
responses, e.g., in terms of serum antibody titer against
the standard antigen (for humoral immunity) and skin test
reaction (for cell-mediated immunity).
ADMINISTRATION
It will be apparent to one of ordinary skill in the
art that the precise amounts of MDP derivative and
antigen needed to produce a given effect will vary with
the particular compounds and antigens, and with the size,
age, and condition of the subject to be treated. Thus,
it is impossible to state exactly the amounts needed:
however, these amounts can easily be determined using
methods known to those of ordinary skill in the art.
The adjuvants and vaccines of the invention are
generally administered by injection, particularly
intramuscular injection, preferably into a large muscle.
In general, an initial vaccination is administered
using the desired antigen and the formulation of the
invention. The vaccination is "boosted" several weeks
8655Y 26310-FF
-28- ~ 33861 8
later (usually 2-6 weeks, for example, 4-6 weeks) using a
vaccine of the invention with or without (preferably
with) the MDP component. Generally, 1-2 mL of a vaccine
(such as are described in the Examples below) is
administered to a human subject in the practice of the
invention.
The following examples are presented as an aid to
those of ordinary skill in the art, and are not to be
considered as a limitation of the invention in any way.
EXAMPLE 1
(Immunogenicity)
(A) Preparation of Formulations:
Adjuvant formulations were prepared as follows for
assay of biological activity. Each emulsion was prepared
at 2X concentration prior to combination with a 2X
solution of antigen.
Formulation 1 (from Allison, U.S. 4,606,918): 5.0%
Pluronic~L121, lOX squalane, 0.4% Tween~80, qs
phosphate buffered saline (pH 7.4); the components
were added to a test tube and vortex-mixed until a
milky emulsion was obtained. This formulation was
prepared immediately prior to administration.
Formulation 2 (Formulation 1 with refrigeration): 5.0%
Pluronic~ L121, 10% squalane, 0.4% Tween~ 80, qs
phosphate buffered saline (pH 7.4); the components
were added to a test tube and vortex-mixed until a
milky emulsion was obtained. The formulation was
then refrigerated at 4C beginning one day prior to
administration.
Formulation 3 (tetra-polyol formulation of the invention):
5.0% Tetronic~ 1501, 10% squalane, 0.4% Tween~
80, qs phosphate buffered saline (pH 7.4); the
components were added to a test tube and
vortex-mixed until a milky emulsion was obtained.
8655Y 26310-FF
~;
-29- l 33 86l 8
Formulation 4 (microfluidized POP-POE adjuvant of the
invention): 5.0% Pluronic~ Ll21, lO~ squalane,
0.4% Tween~ 80, qs phosphate buffered saline (pH
7.4); the components were added to a test tube and
vortex-mixed until a milky emulsion was obtained.
This emulsion was then passed through a
Microfluidizer~ four times. This formulation was
refrigerated with Formulation 2.
To each formulation was then added solid N-acetyl-
muramyl-L-threonyl-D-isoglutamine (Thr-MDP) to a
concentration of 500 ~/mL, to form the complete
adjuvant "concentrate. n The concentrate was then mixed
with a 2X concentration solution of antigen (ovalbumin in
saline, l mg/mL) to form a test vaccine.
(B) ~ioactivity:
Each test vaccine (0.2 mL) was admlnistered to 8
female Guinea pigs. At four weeks following
administration, each animal was boosted with the same
test vaccine (but without the Thr-MDP). Antibody titer
was measured from serum samples collected at weeks 4 and
6 after initial administration. At 6 weeks, each animal
received ovalbumin intradermally, and the diameter of the
erythema, and the infiltration rating were determined
after 24 hours, as an indication of cell-mediated
immunitY.
The results are reported in Tables l and 2. Each
entry represents the mean obtained from 8 animals.
Antibody titers were determined by hemagglutination.
Infiltration was scored visually, on a 1-3 scale (l being
the weakest response, and 3 being a very obvious swelling
at the skin test site).
8655Y 26310-FF
1338~l8
-30-
TABLE 1
Antibody Titer Results
Formulation 4 weeks 6 weeks
1 (control POP-POE) 2.25 + .386.13 + .30
2 (refrigerated POP-POE) 1.38 + .186.50 + .19
3 (tetra-polyol) 4.00 + .388.13 + .30
4 (refrig/~fld POP-POE) 2.87 + .128.00 + .27
TABLE 2
Cell-Mediated Immunity Results
FormulationDiameter Infiltration
(mm)
1 (control pOP-POE) 16.06 1.75
2 (refrigerated POP-POE) 13.81 1.31
3 (tetra-polyol) 19.13 1.94
4 (refrig/~fld POP-POE) 13.56 1.50
The results demonstrate that the tetra-polyol
adjuvants are significantly more effective for increasing
the immunogenicity of antigens, and that the micro-
fluidized POP-POE block polymer adjuvants are at least as
effective as control formulations while demonstrating
superior storage stability.
EXAMPLE 2
(Physical Characteristics)
The formulations prepared in Example l(A) were
examined for physical characteristics.
(A) Separation: Each of the formulations (1-4)
was centrifuged for 30 minutes at 4500 x G, then allowed
to stand to separate into layers. The amount of
separation that occurred was noted and estimated as the
8655Y26310-FF
1 3386 1 ~
-31-
volume percentage of the total occupied by the upper
layer. Formulations 1-3 separated about 10%, while
formulation 4 separated less than about 1%.
(B) Particle Size: Particle size distributions
were analyzed by optical microscopy (Leitz Ortholux II
POL-BK polarized light microscope), transmission electron
microscopy (TEM, using a Hitachi model HS-8-1), and laser
photon correlation spectroscopy (PCS, using a Nicomp
Model 200 laser particle sizer, with a model TC-100
computing autocorrelator). Particle size distributions
were determined for top layers and bottom layers
separately. Samples analyzed by TEM and PCS were diluted
1:100 or greater before analysis. The results
demonstrated that Formulations 1-3 exhibited particle
sizes ranging from < 0.1 ~m to about 25 ~m.
Formulation 4 exhibited particle sizes ranging from
0.1 ~m to about 0.3 ~m (300 nm).
EXAMPLE ~
(Formulations)
Exemplary adjuvant formulations were prepared as
follows:
(A) Tetronic~/Thr-MDP:
Tetronic~ 1501 2.5 9
Squalane 5.0 9
Tween~ 80 0.2 9
Thr-MOP 250.0 mg
Phosphate buffered salineqs to 100.0 mL
The Tetronic~ 1501, squalane, and Tween~ 80 are
placed in an appropriate vessel with 85 mL of phosphate
buffered saline (PBS) and are mixed with a mechanical
mixer (Greerco Homogenizer-Mixer, model #lL-79, Greerco
Corp., Hudson, New Hampshire) at about 4750 rpm for about
30-60 minutes. Then, the Thr-MDP (N-acetylmuramyl-L-
8655Y 26310-FF
1 3386 1 8
-32-
threonyl-D-isoglUtamine) and remaining 15 mL of PBS are
stirred in, producing an adjuvant formulation of the
invention.
(B) Similarly, proceeding as in part (A) above but
substituting N-acetylmuramyl-L- ~aminobutyryl-D-iso-
glutamine (Abu-MDP), 6-0-stearoyl-N-acetylmuramyl-L-
~aminobutyryl-D-isoglutamine (Abu-MDP stearate),
N-acetylmuramyl-L-valyl-D-isoglutamine (Val-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), N-acetyl-
desmethylmuramyl-L-alanyl-D-isoglutamine (desMe-MDP),
N-acetylmuramyl-L-alanyl-D-glutamine butyl ester
(murabutide), n-butyrylmuramyl-L-(~-aminobutyryl)-D-
isoglutamine, and N-acetylmuramyl-L-seryl-D-isoglutamine
(Ser-MDP), for the Thr-MDP, the corresponding adjuvant
formulations are prepared.
(C) Pluronic~/Thr-MDP:
Pluronic~ L121 2.5 9
Squalane 5 0 9
Tween~ 80 0 2 9
Thr-MDP 250.0 mg
Phosphate buffered saline qs to 100.0 mL
The Pluronic~ L121, squalane, and Tween~ 80 are
placed in an appropriate vessel with 85 mL of phosphate
buffered saline (PBS) and are mixed with a mechanical
mixer (e.g., Greerco Homogenizer-Mixer) at about 4750 rpm
for about 5-10 minutes. Then, the Thr-MDP (N-acetyl-
muramyl-L-threonyl-D-isoglutamine) and remaining 15 mL of
PBS are stirred in. The resulting emulsion is then
processed through a Microfluidizer~ (Microfluidics
Corp.) for at least 4 cycles to provide an adjuvant
formulation of the invention. Alternatively, the MDP is
added after the microfluidization step.
(D) Similarly, proceeding as in part (C) above but
substituting N-acetylmuramyl-L- ~aminobutyryl-D-iso-
8655Y 26310-FF
1 3386 1 8
-33-
glutamine (Abu-MDP), 6-0-stearoyl-N-acetylmuramyl-L-
~-aminobutyryl-D-isoglutamine (Abu-MDP stearate),
N-acetylmuramyl-L-valyl-D-isoglutamine (Val-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), N-acetyl-
desmethylmuramyl-L-alanyl-D-isoglutamine (desMe-MDP),
N-acetylmuramyl-L-alanyl-D-glutamine butyl ester
(murabutide), N-butyrylmuramyl-L-( ~aminobutyryl)-D-
isoglutamine, and N-acetylmuramyl-L-seryl-O-isoglutamine
(Ser-MDP), for the Thr-MOP, the corresponding adjuvant
formulations are prepared.
(E) Similarly, the adjuvant formulations prepared
in parts A and B above may be further improved by
microfluidizing as described in parts C and D to form the
corresponding microfluidized Tetronic~ formulations.
(F) Tetronic~/murabutlde:
Tetronic~ 1501 2.0 9
Squalane 6.0 9
Brij~ 80 0.3 9
murabutide 300.0 mg
Phosphate buffered salineqs to 100.0 mL
The formulation is prepared as described in parts A
and E above.
~ G3 Similarly, proceeding as described in parts
A-F above, but substituting Pluronic~ L101 or
Tetronic~ 1301 for Pluronic~ L121 or Tetronic~
1501, the corresponding adJuvants are prepared.
(H) Pluronic~/Ser-MDP concentrate:
Pluronic~ L121 2.5 g
Squalane 5.0 9
Tween~ 80 0.2 9
Ser-MDP 250.0 mg
Phosphate buffered salineqs to 100.0 mL
8655Y 26310-FF
1 3386 18
-~4-
The Pluronic~ L121, squalane, and Tween~ 80 are
placed in an appropriate vessel with PBS (qs to 50 mL)
and are mixed with a mechanical mixer (Greerco
Homogenizer-Mixer) at about 4750 rpm for about 5-10
minutes. The resulting emulsion is then processed
through a Microfluidizer~ for 4 to 10 cycles to provide
an emulsion concentrate. The Ser-MDP and remaining PBS
are provided as a second component to form a
two-component "kit" for extemporaneous preparation of the
adjuvant. To use, the desired amount of antigen is added
to the Ser-MDP solution, and the resulting solution is
mixed vigorously with the emulsion component.
(I) Similarly, proceeding as in part H above but
substituting the components described in parts A-G above,
the corresponding adjuvant kits are prepared.
(J) Vaccines: Vaccines of the invention are
prepared by adding an appropriate amount of antigen to
any of the formulations described above. Suitable
antigens include antigens for hepatitis B, influenza (for
example, A or B), AIDS and herpes. The vaccine may
contain more than one antigen if desired, for example,
antigens for diphtheria, pertussis, and tuberculosis may
be coadministered in a single formulation.
For ease of preparation, a small portion of the PBS
used may be withheld from the adjuvant preparation, e.g.,
one may prepare the adjuvants described above using 90 mL
rather than 100 mL, and use the withheld PBS to
dissolve/suspend the antigen(s). The antigen/PBS
solution is then mixed with the (slightly) concentrated
emulsion to prepare the final vaccine. Alternatively,
and more preferably, an adjuvant emulsion (without MDP)
of two times concentration is mixed with an antigen/MDP
solution of two times concentration.
8655Y 26~10-FF
1 33861 8
ExamDle 4
5ComDarison of Freshl~ Made and Frozen Emulsions
Two times concentrated microfluidized emulsions,
consisting of 10% v/v squalane, 5% v/v Pluronic~ L121 and
0.4% polysorbate 80 in phosphate buffered saline, were used
in the test, having been prepared as for Formulation 4
(Example 1). One emulsion was stored frozen for seven days
before use, while the other was freshly prepared and kept at
room temperature. On the day vaccines were prepared, Thr-MDP
was added to the fresh and thawed emulsions. Equal volumes
of 2X concentrated ovalbumin were added to the 2 lots of
emulsions just before the vaccines were used to ;mml]n;ze
groups of 8 female guinea pigs. The final concentrations of
the constituents of the vaccines were: Phosphate buffered
saline 92.33%; Squalane 5%; Pluronic~ L121, 2.5%; Polysorbate
80, 0.17%; Thr-MDP 250 ~g/ml; and Ovalbumin 1.0 mg/ml.
Guinea pigs were vaccinated on days 0 and 28 with 0.2 ml
of vaccine per ~n;m~l, bled on days 28 and 42, and skin
tested with 10 ~g of ovalbumin on day 42.
The results obtained, as shown below, show that the
efficacy of the frozen material was equivalent to that of the
freshly prepared emulsion.
1 33861 8
-36-
TABLE 1
ANTIBODY TITRESa
28 Days 42 Days
No. of Vehicle Mean Mean Equivalent
Group Animals Preparation Titre + SE Titre + SE Dilutionb
1 8 Fresh 4.6 + 0.2 9.0 + 0.1 18,837
2 8 Frozen 5.1 + 0.3 8.9 + 0.1 17,830
a Titres are expressed as lc93 of the reciprocal of the
serum dilution giving an optical density reading of 0.5
absorbance units, under the conditions of the assay.
b Titre expressed as the reciprocal of the mean serum
dilution.
TABLE 2
DELAYED HYPERSENSITIVITY SKIN REACTIONS
Mean u~ameter ~mm + SE)
No. of Vehicle
Group Animals Preparation24 Hr 48 Hr
1 8 Fresh 14.3 + 0.7 11.0 + 1.7
2 8 Frozen 13.3 + 2.3a 11.4 + 2.ga
a Includes one animal which had no response and may not
have been skin tested.
EXAMPLE 5
Hepatitis Virus Vaccine
Groups of 8 female Hartley guinea pigs were
immunized subcutaneously with a vaccine consisting of
0.5 ~9 or 0.1 ~9 of Hepatitis B virus surface antigen
8655Y 26310-FF
- 37 -
1 33 86 1 8
(HBsAg) in microfluidized adjuvant (prepared as for
Formulation 4, Example 1, without refrigeration) or adsorbed
to alum (commercially available hepatitis vaccine). The
HBsAg in saline and HBsAg adsorbed to alum were provided by
Merck Sharpe and Dohme Research Laboratories. The adjuvant
preparation consisted of 92.33% PBS, 5% squalane, 2.5%
Pluronic L121, 0.17% polysorbate 80, 100 ~g/ml Thr-MDP, and
either 1.0 ~g/ml or 0.2 ~g/ml of HBsAg. Each animal received
0.5 ml of vaccine at day 0 and week 4. The animals were bled
at weeks 4, 6 and 15. Antibody titres were determined by
ELISA techniques and were far superior for the vaccine of the
invention.
TABLE
ANTI-HBsAg TITRES OF POOLED GulN~A PIG SERA
AT 4, 6 OR 15 WEEKS AFTER PRIMARY VACCINATION
E_ISA Titre
HBsAg
Group Vehicle Dose 4 Weeks 6 Weeks 15
(~g) Weeks
1Adjuvant 0.5 814 3409216693
2 Alum 0.5 230 4002 3041
3Adjuvant 0.1 52 6210 4719
4 Alum 0.1 34 1131 1409
~r
- 38 - l 338 6 1 8
ExamDle 6
Influenza Virus Vaccine
MATERIALS AND METHODS
Groups of 10 or 11 6-7 week old female BALE/cJ mice were
;mml]n;zed subcutaneously with the trivalent vaccine prepared
for the 1987-88 influenza season. The influenza virus
strains used were A/Taiwan, A/Leningrad and B/Ann Arbor. The
antigen concentration is expressed in ~g of hemagglutinin
(HA). The vaccine was diluted so that the mice received 0.01
~g of HA of each strain in 0.1 ml of a microfluidized
adjuvant of the invention (2.5% Pluronic~ L121; 5.0%
Sqaulane; 0.17% polysorbate 80; 500 ~g/ml Thr-MDP; (0.1 ~g/ml
15 influenza;) 92.33% PBS; prepared as for Formulation 4,
Example 1, without refrigeration). One group of mice was
given adjuvant (with Thr-MDP) without any influenza antigen.
The groups of mice were ;mml]n; zed as follows:
1. Control - Adjuvant only
2. 0.01 ~g of each strain in Adjuvant
3. 0.01 ~g of each strain in Adjuvant
Groups 1 and 2 were immunized at 0 and 3 weeks while group 3
25 was ;mml]n;zed at 0 time only.
At weeks 3, 5 and 9, 50 ~1 of blood was obtained from
each mouse (under ether anesthesia) via the retro-orbital
plexus. Sera were pooled by group. At week 13 the mice were
30 bled out under ether anesthesia, and sera were kept
individually as well as in pools.
The Table below shows the mean titres determined for
sera from all groups at week 13. For the 0.01 ~g dose
35 levels, there was no significant difference in titre between
the groups given one dose compared to those given 2 doses,
when the anti-A/Taiwan or anti-A/Leningrad titres were
measured. However, one dose induced significantly lower
anti-B/Ann Arbor titres than did 2 doses. (Compare groups 2
and 3).
`.3~
1 33861 8
TABLE
MEAN ANTI-HA TITRES OF MICE
513 WEEKS FOLLOWING IMMUNIZATION
Mean Titre I SE
Boost
HA at
G rou~Dose (,Ug) 3 weeks Vehicle A/Taiwan A/Leningrad B/Ann Arbor
1 0 t Adjuvant <3.0 + ob,c <3.0 +ob <3 0 + ob
2 0.01 1 Adjuvant 9.1 + 0.1 8.0 + 0.2 6.8 + 0.2
3 0.0 1 - Adjuvant 8.8 + 0.2 7.8 + 0.3 5.9 + 0.3
a) Titre is log3 of the reciprocal of the serum
dilution giving an optical density of .05
10absorbance units.
b) Lowest dilution tested was 1/27, i.e., 1/33
c) Sera of 2 animals had titres of 3.1 and
153.2, while for the r~m~;n;ng B sera no
antibody was detectable.
Exam~le 7
The ovalbumin vaccine of Example 4 was prepared as
described in Formulation 4, Example 1, but without the Tween
80.
Exam~le 8
The ovalbumin vaccine of Example 4 was prepared as
described in Formulation 4, Example 1, but the vaccine
composition contained only 1.25% Pluronics~ L121.
Exam~le 9
Other Vaccines
30The ovalbumin vaccine of Example 4 was prepared as
described in Formulation 4, Example 1, but using the
following antigens in place of ovalbumin:
1 3386 1 8
- 40 -
HIV (Human immunodeficiency virus)
Plasmodium yoelii peptides
Influenza viruses (A and B types)
Adenoviruses
Herpes simplex virus type 1, glycoprotein gDl
Melanoma antigens (mouse and human)
Foot and mouth disease virus
Hepatits B virus surface antigens
Hepatitis A virus
Para-influenza 3 glycoproteins
SIV (simian immunodeficiency virus)
Shistoma mansoni cercaria
Folate hydrolease
Polio virus
Mouse idiotype antibody
Bacterial toxoids
Human tumor associated antigens
Simian retrovirus (type 1 & 2) peptides
Type D retrovirus
Parasite antigens
LHRH
Mouse IgG peptides
Brucella abortus proteins
HIV proteins
Fibroblast growth factors (a and ~)
IL-6
Herpes simplex virus, type 2, early gene 22
Feline leukemia virus
[The weight of MDP in the vaccine was subject to minor
variation depending on the species of animal tested. In some
cases the emulsion was not refrigerated before addition of
the antigen and MDP.]
- 40a - l 33861 8
Exam~le 10
An ovalbumin vaccine in microfluidized adjuvant
consisting of:
2.5% Tetronic 1501
5% Squalane
0.2% Tween 80
Phosphate buffered saline to volume
250 ~g/ml Thr-MDP
Ovalbumin
was prepared as for Formulation 4, Example 1, without
refrigeration.
A group of 8 female Sim:(HA) guinea pigs, 350-400 g, was
used. The animals were injected sc in the nuchal region with
0.2 ml of the formulation; each animal received 200 ~g OA and
50 ~g Thr-MDP on Day 0, and 50 ~g OA and 50 ~g Thr-MDP on Day
28. They were bled by cardiac puncture on Days 28 and 42,
and skin tested on Day 42 with 10 ~g OA, given id. The
diameter and induration of the skin tests were measured 24
and 42 hours later. The antibody titres were determined by
passive hemagglutination and by ELISA (see below).
ANTIBODY TITRES - EL-SA METHOD
28 Days 42 Days
Titre + SE Titre + SE Actual Dilution
5.26 + 0.25 9.46 + 0.10 32,626
- 40b - l 3386 1 8
DELAYED HYPERSENSITIVITY
Mean Diameter I No. Inf. Inf. Score
SE
24 Hr 48 Hr 24 Hr 48 Hr 24 Hr 48 Hr
16.78 + 0.64 12.00 + 0.49 5/7 4/7 1.50 1.36
ExamDle 11
In the tests reported above, no significant side effects
were observed.
.
